Cocaine addiction is a vexing public health problem for which no effective pharmacotherapies have been developed. Naturally derived bioactive compounds have been a rich source for modern drug development. Under our CERC program project Alternative Therapies for Alcohol and Drug Abuse (P01-AT- 002038) funded by NCCAM, eight isoquinoline alkaloids, including L-tetrahydropalmatine (L-THP), were isolated and characterized from tubers of Corydalis yanhuso, which is one of the five components of an herbal remedy (NPI-025) used in China to treat opium smoking for over 150 years. Interestingly, L-THP has been used clinically in China for more than 30 years as an analgesic with sedative/hypnotic properties and recently to treat heroin addiction. In animal models, L-THP reduced reinforcing property of cocaine and reinstatement of cocaine seeking following withdrawal. Importantly, L-THP itself had no rewarding effect, suggesting absence of abuse liability. Recently, Dr. Jia-Bei Wang of the University of Maryland received an NIH grant to conduct a clinical trial on L-THP for treatment of cocaine addiction in the US. Despite its long history of clinical use, the mechanisms of action of L-THP are not clear. Although some in vivo effects appear to be mediated by dopamine receptors, we found that L-THP had low or no affinity for the five subtypes (D1- D5) of DA receptors (see C.1.2). In addition, it did not bind t about 60 neurotransmitter receptors, ion channels, and transporters involved in pain modulation and drug addiction that we screened (see C.1.2). Following L-THP administration in mice or rats, we identified in serum the mono-demethylated analogs L- corypalmine (L-CP) and L-corydalmine (L-CD) to be the major metabolites and L-isocorypalmine (L-ICP) to be a minor metabolite (see C.1.3). We also found that L-ICP had moderate to high affinity for DA receptors (see C.1.4) and reduced cocaine-induced hyperlocomotion, behavioral sensitization and conditioned place preference (See C.1.5-1.8). Thus, we hypothesize that L-THP is a prodrug and its effects are due to demethylated metabolites acting on multiple DA receptors to modulate cocaine-induced behaviors. Consequently, we propose to further pharmacologically characterize L-THP and its demethylated metabolites to gain a better understanding of the mechanisms of action. Our specific aims are to (1) prepare L-THP and chemically synthesize its metabolites in sufficient quantity for pharmacological studies; (2) determine the metabolism and pharmacokinetic profiles of L-THP; (3) identify pharmacological activity of major metabolites at DA receptors and other targets; and (4) examine the effects of L-THP and its metabolites on cocaine self administration and reinstatement of drug seeking following withdrawal. The proposed research will enhance the understanding of mechanisms of action of L-THP, which may open a new avenue for mechanism-based drug discovery for cocaine addiction. As one of the major side effects of L-THP is sedation, our proposed study may yield candidate drugs with less sedation, as we have demonstrated for L-ICP. PUBLIC HEALTH RELEVANCE: The overall objective is to study the mechanism of action of the natural product L- tetrahydropalmatine (L-THP) as an alternative therapy against cocaine dependence, for which there is no satisfactory pharmacotherapy. Studies in our laboratory and others have shown that L-THP reduced cocaine self- administration, reinstatement of cocaine seeking, cocaine-induced conditioned place preference (CPP), and brain-stimulation enhancement, while having no intrinsic rewarding effect, which suggests absence of abuse liability. Consequently, we propose to further pharmacologically characterize its demethylated metabolites to gain a better understanding of the mechanism of action and to support our hypothesis that it is a prodrug and its effects are due to demethylated metabolites acting on multiple DA receptors to modulate cocaine-induced behaviors and neurochemical changes.